Germination of spores

ABSTRACT

THIS INVENTION RELATES TO METHODS AND COMPOSITIONS FOR GERMINATING SPORES OF THE BACILLUS OR CLOSTRIDIUM GENERA ULTILIZING LIPASES.

United States Patent Oflice 3,672,956 Patented June 27, 1972 3,672,956GERMINATION F SPORES Lewis G. Scharpf, Jr., Kirkwood, Mo., assignor toMonsanto Company, St. Louis, M0. N0 Drawing. Filed Jan. 30, 1970, Ser.No. 7,264

Int. Cl. C12b 1/00 U.S. Cl. 195-96 17 Claims ABSTRACT OF THE DISCLOSUREThis invention relates to methods and compositions for germinatingspores of the Bacillus or Clostridium genera utilizing lipases.

BACKGROUND OF THE INVENTION Field of the invention This inventionrelates to the use of enzymes to germinate bacterial spores, morespecifically, this invention relates to the use of lipases to germinatesuch spores and to compositions including such enzymes.

Description of the prior art Germination is described as an irreversibleprocess in which a number of events take place shortly after exposure ofspores to specific stimulants. During this period of time, referred toas the germination phase, the spore loses heat resistance, dipicolinicacid, impermeability to dyes, calcium, refractility and optical densityto visible light. The spore is now referred to as a germinated form or agerminated spore. However, all of the above mentioned changes do nottake place at the same rate, for example, the complete germination phasefor a spore may take two hours, but it may take only one hour for thespore to be come heat sensitive whereas it may take two hours for thespore to lose dipicolinic acid. Outgrowth is described as the period ofdevelopment after the germination phase until the beginning of the firstcell division. During this period of time, referred to as the outgrowthphase, the spore core membrane becomes a cell wall of the emergingvegetative cell which then elongates and divides. Vegetative cellssporulate and then the life cycle is repeated.

It is well-known that the vegetative cells from Bacillus or Clostridiumspores are responsible for food spoilage including flat sour spoilageand thermophilic anoerobic spoilage. For example, various saladdressings, vegetables and canned milk are spoiled by B. subtilisvegetative cells; likewise, dairy foods are spoiled by B. megaterium andfruits and vegetables by B. stearothermophilus. In order to destroy thenon-germinated form of the spores present in food, as is well-known,they must be heated at high temperatures for extended periods of time.By destroying these spores under such severe conditions, generallyflavor, texture and vitamin content of the food is adversely affected.One way of reducing these adverse effects is to first germinate thespores to make them heat sensitive so that these forms may be easilydestroyed by heating at lower temperatures for short periods of time.

It is known that some surface active agents, amines, alkanes or metalchelates induce germination and decrease the period of time necessaryfor the germination phase, however, some cannot be used to germinatespores to prevent food spoilage because of toxicity characteristics orbecause they impart undesirable flavor characteristics to the food.Additionally, it is disclosed in US. Pat. 3,276,840 that a proteolyticenzyme extracted from bacterial spores of the Bacillus or Clostridiumgenera such as B. subtilis, for example, can be used directly to inducegermination of bacterial spores of the Bacillus and Clostridium generasuch as B. subtilis, B. cereus and B. lichenformis. However, it is atime consuming and difficult process to extract the protease enzymesbefore use, consequently, there may not be a sufiicient supply of theenzymes which could be used on a large scale in the food industry. Also,these proteases at such levels might act on and hydrolyze suchsubstrates as meat, milk, poultry, eggs and other proteins to produceundesirable effects and flavor texture of the final product.

It would, therefore, be an advancement in the art to provide a methodfor the germination of Bacillus and Clostridium spores which decreasesthe period of time required for the germination phase and which can beadvantageously utilized in food preservation with none of the abovementioned disadvantages.

SUMMARY OF THE INVENTION Briefly, in accordance with this invention, itwas found that a lipase enzyme may be used directly to inducegermination of bacterial spores of Bacillus and Clostridium genera, suchas Bacillus subtilis, Bacillus mega'terium, Bacillus stearothermophilus,for example, with corresponding decreases in the time required for thegermination phase.

In another embodiment of this invention, the time re quired for thegermination phase is further decreased by combining a lipase and eithera physiological germinant, for example, amino acids or sugars or asupplemental enzyme hereinafter set forth or both. In still anotherembodiment of this invention, a process is provided where the spores arefirst germinated and then the germinated forms are treated with heat orionizing radiation such as gamma radiation or high energy electrons todestroy these germinated forms of the spores. Finally, in anotherembodiment of this invention, a process is provided wherein the sporesbefore germination are treated with sublethal heat to decrease the timerequired for the germination phase of such spores.

By utilizing one of the processes in accordance with this invention, thespores of the Bacillus or Clostridium genera are germinated with acorresponding decrease in the time needed for the germination phase.Additionally, the spores become heat sensitive at the same rate orbefore many of the other mentioned characteristic charges ofgermination. This is a substantial advantage in food processing, for assoon as the spores become heat sensitive, they may be destroyed, whichdecreases the overall processing time required for food. Anotherunexpectant advantage found using one of the processes of this inventionwas that a percentage of the spores undergoing germination were renderednot viable, i.e., not able to continue their development in theoutgrowth phase. This is another substantial advantage in foodprocesses, as in some cases, further preservation steps may not beneeded. The lipases advantageously utilized in accordance with one ofthe processes of this invention are readily available in large supplies.Such enzymes have none of the nudesirable side effects such as toxicityand therefore can be used in preserving food. Also, such enzymecombinations will not produce undesirable side effects generallyencountered by using large quantities of proteases. When the processesof this invention are utilized to germinate the spores and subsequentlyto destroy them in preserving food, the vitamin retention texture andflavor characteristics is generally not adversely alfected.

Additionally, it is known that members from the Bacillus genera causeundesirable effects such as the formation of slime in high octanehydrocarbon fuels which may lead to the malfunctioning of combustion jetengines and subsequently to disruption of the aircraft using ahydrocarbon fuel in which such a slime has formed. By utilizing theprocess of this invention, spores of the Bacillus genera may begerminated and this germinated form easily destroyed by heating at lowtemperatures. Consequently, the problem of slime may be substantiallyreduced or eliminated.

DETAILED DESCRIPTION OF THE INVENTION As mentioned hereinbefore in thepractice of this invention, the germinating agents which may be employedare lipase or a combination of lipase with either a physiologicalgerminant or supplemental enzymes or both.

Lipases that are utilized according to the invention may be obtainedfrom plants, animals or micro-organisms. Generally, it is preferred touse lipases obtained from micro-organisms as such lipases are readilyavailable from said micro-organisms by using standard fermentationtechniques.

The micro-organisms that may be used to supply the lipases may be of afungal origin or bacterial origin. Those of fungal origin includeAspergillus and Candida and Rhizopus. Examples of particular speciesinclude Candida cylindracea, Rhizopus delemar, Mycotor'ula lipolytica,Deotricum candidum, Aspergillus niger and Aspergillus oryzae. Those ofbacterial origin include Pseudomonas, with Pseudomonas fragii being aspecific species.

The actual amounts of lipase which are used to germinate spores,according to the practice of this invention, will depend, to someextent, upon the spores being germinated. In most instances, theeffective concentration. is such that the lipase is present in an amountto provide about units of activity per 10 to 10 spores. Lipase presentin an amount to provide less than 10 units of activity was found not tosignificantly decrease the time needed for the germination phase. Thereis no upper limit on the amount of lipase that maybe present to decreasethe time required for the germination phase, except as dictated bypracticalities of cost and flavor, for example, an amount of lipasepresent to provide 100,000 units of activity per 10 to 10 spores may besuitably employed. It is preferred in most instances, that the lipase bepresent to provide about 800 units of activity to about 30,000 units ofactivity per 10 to 10 spores. A unit of lipase is defined as the amountwhich can liberate 1 mole of fatty acid per minute from olive oil at 37C., pH 7.

The procedure for determining this activity is described in I. Agr.Chemical Society, Japan 36, 860-864 (1962).

It should be noted that as the concentration of spores increase, theamount of lipase necessary for germination generally does notnecessarily increase. It was found that if 10 spores of 10 spores arepresent, the time required for the germination phase will besubstantially the same in both instances utilizing about 2,000 units ofactivity of lipase.

These physiological germinants include sugars, amino acids andnucleocides. Examples of sugars include glucose, maltose, lactose andsucrose. Examples of amino acids include tyrosine, L-alanine, DL-valine,DL-cysteine, methionine, glutamic acid, L-arginine, L-phenylalanine,L-leucine, L-tryptophane, aspartic acid, glycine, lysine, L-isoleucine,histidine, serine, threonine, and proline. Examples of nucleosidesinclude inosine, guanosine and adenosine. Glucose, L-alanine and inosineare preferred.

Some of these physiological germinants are more specific for germinatingcertain spores and depending on the spore to be germinated, faster timesfor the germination phase are obtained. or example, the spores of B.subtilis and B. stearothermophilus are germinated faster employingglucose or L-alanine or a combination thereof. However, in the case ofB. megaterium spores, a faster germination time is obtained employingthe physiological germinant inosine.

The amount of the physiological germinant utilized according to thisinvention will depend to some extent, upon the particular germinant andthe spores to be germinated. An effective concentration of thephysiological germinant is at least .001 mg. per 10 to 10 spores. Inmost instances, it was found that an amount below .001 mg. does notsufliciently decrease the time required for the germination phase.Generally, the preferred range is from about 0.01 mg. to about 10 mg.per 10 to 10 spores. Although the physiological germinants may be usedin amounts above 10 mg., for example 25 mg., usually it is withoutcommensurate advantage. In the case of L-alanine, for example, an amountof about 2 to about 5 milligrams/l0 to about 10 spores is especiallypreferred. In the case of glucose and inosine, about 0.05 to about 0.4mg/l0 to 10 spores is especially preferred.

By combining lipase with supplementary enzymes to germinate spores, thetime required for the germination phase is decreased. These include thewell-known enzymes such as proteases, amylases, phosphatases or mixturesof the foregoing.

These enzymes useful in this invention may be obtained from animals,plants or micro-organisms. It is preferred to use enzymatically activesubstances of a microbial origin, and more preferred to use those of abacterial origin as they can be economically produced in appreciableamounts. These bacteria produce either a single enzyme or a mixture ofenzymes.

Examples include Bacillus, Aspergillus or Streptomyces micro-organismsincluding various B. subtilis strains such as B. subtilis strain NRRLB-3411 (U.S. Department of Agriculture collection, Peoria, Ill.) B.subtilis strain NRRL 644, B. subtilis strain LAM 1523 (Japanese CultureCollection) all of which produce a mixture of proteases and amylase.Other organisms include B. thermoproteolyticus, Streptomyces griseus,Aspergillus oryzae, Streptomyces rectus, Streptomyces naraensis, and B.subtilis var amylosacchariticus, all of which produce either a mixtureof protease and amylase or only neutral protease. Streptomyces griseusstrain K-l produces a predominantly neutral protease.

Neutral protease as used in this application refers to a metallo-enzymewhich has its optimum activity at a pH of about 6 to about 8, isinhibited by metal-chelating agents but unaffected by such inhibitors asdi-isopropyl fluoro phosphate (DFP) and hydrolyses substrates such asfurylacryloylglycyl-L-leucine amide (FAGLA), but does not possessactivity against esters such as p-nitrophenyl acetate of N-CBZ-glycinep-nitro-phenyl ester. A metallo enzyme is one containing metal essentialfor activity. Alkaline protease as used in this application refers to anenzyme which has its optimum activity at a pH of about 8 to about 11, isinhibited by DFP, but not by metal chelating agents and possessesactivity against esters such as N-CBZ-glycine p-nitro-phenyl ester, butnot against FAGLA.

A particularly good source of the enzymatically active substance is anenzyme mixture produced by Bacillus subtilis strain NRRL B-341l. Aprocess for producing this organism and enzyme therefrom is described inUS. Pat. 3,031,380. The enzymatically active substance produced by thisorganism has been found generally to consist of two proteases, neutralprotease, alkaline protease, and amylase. There are generally about 700thousand to about 2 million casein units of neutral protease activityper gram of isolated solids and about 250 thousand to about 500 thousandcasein units of alkaline protease activity per gram as determined by acasein digestion technique (hereinafter to be described). There areabout 300 thousand to about 500 thousand units of amylase as determinedby the Bernfeld method (hereinafter to be described). As pointed out inthe cited patent, the relative proportions of protease to amylase willvary depending on the exact conditions of growth of the micro-organism,but it has been found that the neutral and alkaline protease and theamylase will be produced, in at least some amounts, almost regardless ofchanges in the culture medium and other conditions of growth of themicroorganism.

Various analytical methods are available for determining enzymeactivity, for example, protease activity can be determined by well-knownprotein digestion methods using protein substrates such as casein,hemoglobin, bovine serum albumin or gelatin. According to such tests, aprotease catalyzes the hydrolysis of a protein (for example, casein) fora certain period of time under controlled conditions of temperature, pHand substrate concentration; the reaction is stopped by addition oftrichloroacetic acid, and the solution filtered. The solubilizedfragments in the filtrate are determined either by measurement ofabsorbance in the ultraviolet range or rendered visible by reaction withFolin phenol reagent, and absorbance measured in the visible range andenzyme activity expressed in terms of tyrosine equivalents. This methodis more fully described in the Journal of General Physiology, 30, (1947)291 and in Methods of Enzymology, 2, New York: Academic Press 1955, 33.

In this application when neutral protease activity is expressed incasein units, it is understood that such activity is determined at pH 7,and when alkaline protease is expressed in casein units, it isunderstood that such activity is determined at pH 10.

Other methods for determining protease activity make use of lowmolecular weight substrates in spectrophotometric assays, for example,the substrate FAGLA is specific for neutral protease and is used todetermine neutral protease activity as described in BiochemicalBiophysical Research Communications, 32, 326 (1968).

Amylase activity is generally determined by the wellknoWndinitrosalicyclic acid method of Bernfeld as described in Methods ofEnzymology, Academic Press, 1955, II p. 949. According to this test,amylase catalyzes the hydrolysis of the starch to reducing sugar at agiven time and temperature. The reaction is stopped and color developedby the addition of dinitrosalicyclic acid. The optical density of thesolution is estimated from a standard curve prepared with known amountsof maltose hydrate. In this application, when units of activity ofamylase are stated, it is understood that the Bernfeld technique isemployed to determine such activity.

The neutral proteases as a group possess different specificity from thealkaline proteases as a group. For example, alkaline proteases possessesterase activity due apparently to their mechanism of action and not totheir pH optimum while neutral proteases do not. Tests demonstratingthis fact are more fully described in Arch. Biochem. Biophys. 123,(1968) 572. Various techniques can be utilized to separate differentcomponents of mixtures, for example, neutral protease may be separatedfrom enzyme mixtures, by ion exchange chromotography as described in theJournal of Biological Chemistry, 239, (1964, 3706) and in Agr. Biol.Chem. 30, (1966) 651.

Alkaline phosphatase has its activity between about pH 7 and pH 11 at 25C. It can be obtained from the calf intestinal mucosa. It is alsoproduced by E. coli. One unit of activity of alkaline phosphatase isdefined as that which will hydrolyze one mole of p-nitro-phenylphosphate in 1 minute at a pH of 10.4 and at a temperature of 37 C.

It is preferred to use alkaline protease or a-amylase.

It should be noted that some of these enzymes are specific forgerminating certain types of spores, for example, alkaline protease whencombined with lipase gives a faster germination rate for germination ofB. megaterium and B. stearothermophilus spores.

The amounts of supplemental enzyme which can be advantageously utilizedaccording to this invention with lipase will depend to some extent uponthe particular enzymes and the spores to be germinated. Satisfactoryresults are obtained, in most instances, in the case of alkalineprotease when the effective concentration is such that as little asunits of activity are present, in the case of neutral protease when theeffective concentration is such that as little as 100 units of activityare present, in the case of amylase when the effective concentration issuch that as little as 500 units of activity are present, and in thecase of alkaline phosphatase when the effective concentration is suchthat as little as 200 units of activity are present, all effectiveconcentrations per 10 to 10 spores. Generally, there is no upper limiton the amount of supplemental enzymes that may be utilized except asnecessitated by matters of cost and flavor. For example, satisfactoryresults are obtained when alkaline protease is present in an amount toprovide about 1,000,000 units of activity, in the case of neutralprotease when it is present in an amount to provide at least 1,000,000units of activity, in the case of amylase when it is present to provideabout 5,000,000 units of activity and in the case of alkalinephosphatase when it is present to provide about 1,000 units of activityper 10 to 10 spores. It is preferred to use alkaline protease in anamount to provide about 10,000 to about 500,000 units of activity per 10to 10 spores. It is preferred that neutral protease be present in anamount of from about 10,000 units to about 500,000 units per 10 to 10spores. In the case of amylase, it is preferred that it be present in anamount to provide from about 50,000 units of activity to about 1,000,000units of activity per 10 to 10 spores. It is preferred that alkalinephosphatase be present in an amount to provide about 200 units ofactivity to about 600 units of activity per 10 to 10 spores.

As mentioned hereinbefore, another embodiment of this invention is acombination of lipase with either a physiological germinant or asupplementary enzyme or both. The compositions may contain any of thehereinbefore mentioned enzymes or a physiological germinant in additionto the lipase.

The compositions contain the components in the effective germinatingconcentrations hereinbefore mentioned. For example, a composition wherelipase is combined with a physiological germinant would contain aneffective concentration of the lipase and an effective concentration ofthe physiological germinant hereinbefore mentioned. When the mixturecontained lipase and supplementary enzymes, the composition wouldcontain an effective concentration of the lipase and an effectiveconcentration of the supplemental enzymes pointed out hereinbefore. Whenthe compositions contain the lipase, a physiological germinant andsupplemental enzymes, the composition would contain these components inthe effective concentrations set forth herein.

Preferred compositions include (1) lipase present in an amount toprovide about 10,000 units to about 500,000 units of activity per gramof composition, and inosine in an amount of about 100 mg. to about 200mg. per gram of composition, (2) lipase present in an amount to prw videabout 10,000 units of activity to about 500,000 units of activity pergram of composition, inosine in an amount of about 100 mg. to about 200mg, per gram of composition, and alkaline protease present in an amountto provide about 800,000 units of activity to about 1,200,000 units ofactivity per gram of the composition, (3) lipase present in an amount toprovide about 10,000 units of activity to about 500,000 units ofactivity per gram of the composition, and glucose in an amount of fromabout 100 mg. to about 200 mg. per gram of the composition and (4)lipase present in an amount to provide about 10,000 units of activity toabout 500,000 units of activity, per gram of the composition, glucose inan amount of about 100 mg. to about 200 mg., per gram of the compositionand alkaline protease present in an amount to provide about 800,000units of activity to about 1,200,000 units of activity. Compositions (1)and (2) are especially elfective in germinating B. megaterium spores.Compositions (3) and (4) are especially effective in germinating PROCESSAs mentioned hereinbefore, the spores of the Bacillus or Clostridiumgenera may be germinated in any environment by bringing the lipase incontact with the spores. In most instances, however, the enzyme isbrought into contact with the spores in the presence of some moisture.The amount of moisture is not critical, for example, that found inordinary vegetables such as carrots would be sufficient to bring aboutthe germination in accordance with this invention, on the other hand,the spores could be germinated in the presence of more water, forexample, at least 50%. Likewise, when the spores are germinated by alipase in combination with a physiological germinant or supplementaryenzyme or both, the components may be added separately or may be addedas one of the novel compositions containing all of the componentshereinbefore mentioned.

It was unexpectedly found, however, that by first pretreating the sporeswith one germinating agent for a period of time then adding another,that better germination rates were obtained than by using either alone,as using them simultaneously. Generally, the spores are treated with onecomponent for a period of about 10 minutes to about 60 minutes,preferably about 20 minutes to about 45 minutes at a temperature ofabout 25 to about 50 C., preferably from about 35 to about 45 C. Thisprocedure is very effective in treating B. megaterium spores. The sporesare pretreated with lipase for about 30 minutes then with alkalineprotease.

When the spores are germinated in the presence of moisture, generallythe pH of such aqueous medium is from about 5 to about 10, preferablyfrom about 7 to about 9. This pH can readily be maintained by using anyof the normal buffering agents such as the alkali metal phosphatesincluding sodium phosphate, potassium phosphate or any combinationthereof.

Generally, in the practice of this invention, the operable range for thetemperature is from about -10 C. to about 100 C., preferably from about25 C. to about 50 C., more preferably from about 35 C. to about 45 C.

The period of time the lipases or lipase combinations should be incontact with the spores to bring about germination will depend to someextent upon the spores being germinated and the particular combinationif such is utilized. The lipase or the lipase combination should be incontact with the spores for a sufficient time to induce germination. Asmentioned hereinbefore, the period of time that the lipase combinationmust be in contact with the spores is shorter because these combinationsbring about faster germination of the spores. In most instances, a sufiicient contact time is at least 5 minutes. As a general rule, it has beenfound that if the contact time is below 5 minutes, the time required forthe germination phase will not be appreciably reduced. Usually, the'contact time is from about minutes to about 12 hours. In the case whenlipase is used as the germinating agent, the contact time, in mostinstances, is about 2 hours to about 6 hours. When lipase is combinedwith a physiological germinant, generally the contact time is from about30 minutes to about 60 minutes. When lipase is combined with anotherenzyme, generally, the contact time is from about minutes to about 50minutes. When lipase is combined with another enzyme and a physiologicalgerminant, the contact time, in most instances is from about 15 minutesto about 30 minutes. Although the contact time may exceed 12 hours, evenup to 36 hours, there is no appreciable increase in the extent ofgermination of the spores.

Once the spores have been germinated using one of the above-mentionedgerminating agents, the germinated form may be destroyed utilizing heatand ionizing radiation, for example, which comes from gamma radiation orhigh energy electrons. Generally, these germinated forms of the sporesare heated at a sufiicient temperature and for a sufficient period oftime to destroy such forms. In most instances, the temperature must beat least 5 C. it was found that when temperatures below 5 C. wereemployed, that the germinated forms of the spores were not destroyed. Anoperable temperature range is from about 20 C. to about 120 C.,preferably from about 65 C. to about C. Although temperatures above C.may be employed to destroy the germinated spore forms even up to 150 C.,this is Without any added advantage. Generally, the spores are heatedfor a time period of at least 30 seconds. In most instances, it wasfound that a time period below 30 seconds was not a sufiicient time todestroy a great majority of the spore forms. Generally, the germinatedspore forms are heated for a period of 1 minute to about 90 minutes, andpreferably from about 15 minutes to about 30 minutes. Although the sporecan be heated for a period of time above 90 minutes, even up to 180minutes, generally, this is without any added advantage.

In another embodiment of this invention, the spores may be firstpreheated with subleathal heat followed by enzyme treatment. This isheat below that which is required to destroy such spores. The period oftime and the temperature, depends to some extent, upon the spores.Generally, in most instances, the temperature is from about 40 C. toabout C., preferably from about 65 C. to about 100 C., and the period oftime is from about 30 seconds to about 60 minutes, preferably from about10 minutes to about 20 minutes.

The following examples are intended to illustrate the present inventionbut not to limit the scope thereof.

EXAMPLE I Preparation of B. megalerium. spores A culture of B.megaterium is rehydrated, then plated out on 1% casein agar andincubated at 37 C. for 24 hours. A transfer from a colony on this plateis made to a trypto case soy agar slant containing 0.5% glucose.

The culture from this slant is transferred into ml. of nutrient broth asdescribed by Rode and Foster Proc. Nat. Acad. Sci., US. 46,118, 1960,and having the following formula:

A 500 ml. Erlenmeyer flask containing the culture and the broth isplaced on a rotary shaker at 300 r.p.m. for 24 hours. Sterile nutrientagar 100 x 15 mm. plates are prepared and inoculated with 0.5-1.0 ml. ofactive inoculum from the flask and incubated at 3 C. for 3 days duringwhich time sporulation takes place.

HARVESTING THE SPORES After sporulation is complete, autolysis iseffected by keeping the plates at 4 C. for three days. The plates areflooded with chilled, sterile deionized water to suspend the spores andthe spores are collected by centrifugation at 3 C. The spores areresuspended in sterile (millipore filtered) lysozyme solution containing0.5 ml. per milliliter of lysozyme. The temperature is maintained at 37C. and agitation is carried out for 2 hours on a magnetic stirrer. ThepH of the suspension is raised to pH 10 to effect further solubilizationof vegetative cells. After 5 minutes, the pH is readjusted back to pH 7.The spores are again collected and washed from 6 to 8 times with steriledeionized water. Concentrated spore suspensions are stored in glassbottles at 3 C. Spore suspensions are free of non-spore fragments andother debris and contain less than 3% of germinated forms. The sporeconcentration is determined by a dilution of the heat activatedsuspension on nutrient agar. Colonies are counted after 24 hours at 30C. The spore concentration is 10 spores/ml. of suspension.

Preparation of B. subtilis spores A slant culture of B. subtilis(ATCC7953) is innoculated into 1 liter Roux bottles containing 250 ml.of nutrient agar at a pH of 7.4 supplemented with 0.01 mg./ml. of MnSO-1H 'O and 8 mg./ml. of NaCl. These bottles were held at 30 C. for 3days.

HARVESTING OF SPORES The spores are harvested according to the procedureset forth in Example I. Spore concentration is determined by plating adilution of heat-activated suspensions. Colonies are counted after 24hours at 30 C. The spores are plated out on nutrient agar.

DETERMINATION OF GERMINATION TIMES A 10 milliliter aqueous suspension ofthe spores of Bacillus megaterium having a spore concentration of l0/ml. and a pH of about 8.0 buttered with 0.1 M sodium phosphate and a 10milliliter suspension of Bacillus subtilis having a sport concentrationof 10 /ml. and a pH of about 8.0 buffered with 0.1 M sodium phosphateare prepared. 100 mg. of lipase containing about 2,000 units of lipaseis added to each of the suspensions of spores.

The time required for the germination phase measured at 37 C. isfollowed by measuring the optical density at 660 m in a Bausch and Lombspectronic 20, spectrophotometer at various time intervals. These dataare plotted as the ratio of O.D. /O.D. against time where O.D. equaloptical density at time T and O.D. equal optical density initial. Theperiod of time required for the enzyme to produce a 50% reduction inoptical density is determined.

Germination is also determined by counting the number of refractile andphase dark spores at intervals using the phase contrast microscope.

The above procedure was followed and the half-times given in Table I.The half-time was also determined for a 10 ml. spore suspension of B.megaterium without lipase and for B. substilis without lipase.

TABLE I Half times in minutes Without lipase With lipasefiilhlliffiffkIZi: Z238 A microscopic examination also showed that about50% of the spores in each instance had been germinated.

EXAMPLE II 10 is obtained from B. subtilis. Following the aboveprocedures, the half-times set forth in Table 2 were obtained.

TABLE 2 Half time in minutes Without With Lipase and lipase lipasealkaline (Table I) (Table I) portease B. megaterium 500 331. 2 18 B.subtilis 500 156 155 As can be seen from the above table, when lipaseand alkaline protease are added to the spores, the time required forgermination is still further decreased.

EXAMPLE 1H Preparation of Bacillus stearothermophilis spores A slantculture of B. stearothermophilus is transferred to m1. of nutrient brothin a 500 ml. Erlenmeyer flask. The flask is placed on a rotary shaker at300 r.p.m. for 24 hours. Sterile nutrient agar 100 x 15 mm. plates areinnoculated with 0.5-l.0 mls. of the contents of the flask and held for7 days at 55 C. during which time the organisms sporulate.

HARVESTING THE SPORES The spores are harvested in the same manner asthat for B. megaterium spores. The spore concentration is 10 spores/ml.of solution.

DETERMINATION OF GERMINATION TIMES TABLE 3 Half-time in minutesButter-physio- Bufier-physiological logical germigerminant nant pluslipase B. megaterium 200 49 B. stearothermophilus 500 156 A microscopicexamination also showed that about 50% of the spores in each instancehad been germinated.

As can be seen from the above table, the presence of physiologicalgerminants decreased the time for the germination phase.

EXAMPLE IV The procedure of Example III is repeated on B. megateriumwith the exception that for 200 mg. lipase in one instance (a) 100 mg.of lipase containing 2,000 units of lipase and 40 mg. of alkalineprotease containing 44,000 units of activity are simultaneously added tothe spores. The alkaline protease is obtained from B. subtilis. Inanother instance, (b) 100 mg. of lipase having the same activity asabove is added to the spores and allowed to contact the spores for 30minutes at 37 C. Then 40 mg. of alkaline protease having the sameactivity as above is added. In another instance (c) 0.5 ml. of water iscontacted with the spores for 30 minutes at 37 C. Then the alkalineprotease is added. In still another instance (d), 40 mg. of alkalineprotease containing 44,000 units of 11 activity are contacted with thespores. Following the above precedures, the half-times set forth inTable 4 were obtained.

TABLE 4 Instance: Half-time in minutes (a) Simultaneous addition 33.7(b) Pretreatment with lipase 19.8 (c) Pretreatment with water 44.5 ((1)Alkaline protease alone 45.9 (e) Lipase alone (Example III) 49.0

As can be seen from the above table, when lipase and alkaline proteaseare added to the spores, germination phase takes place in less time thaneither alone. Also pretreatment by lipase, then addition of alkalineprotease gives unexpectedly even better results.

EXAMPLE V The viability of B. megaterium and B. subtilis spores isdetermined by repeating the procedure of Example III with the exceptionthat B. subtilis spores are substituted for B. stearorhermophilusspores. After the lipase is in contact with B. subtilis spores forvarious time intervals survivors are determined by plating in triplicatesuitable dilutions of the spores suspensions. 2% nutrient agarsupplemented with 0.1% starch is used to determine the viability of B.megaterium cells to form colonies and nutrient agar is used for B.subtilis cells. The plates are incubated for 16 to 24 hours at 37 C. Thecolonies are counted according to plate count techniques and percentviability determined.

In determining the heat resistance, the procedure of Example III isfollowed with the exception that B. subtilis spores are substituted forB. stearothermophilus spores. After the lipase is in contact with B.subtilis spores for various time intervals, heat resistance isdetermined by heating the germinated spores at 85 C. for 15 minutes. Inthe case of B. mega'terium', the germinated spores are heated at atemperature of 65 C. for 20 minutes. After the B. subtilis spores areheated, suitable dilutions of the suspension of heated spores are platedon 2% nutrient agar and incubated for 16 to 24 hours at a temperature of37 C. In the case of B. megaterium, 2% nutrient agar supplemented with0.1% starch is used for plating. The colonies are counted according tostandard plate count techniques and percent survivors determined therebyindicating heat resistance.

By using the procedure set forth, the following results were obtained.

TABLE 5 Viability, percent Heat-resistance, percent survivors survivorsAlkaline Alkaline protease protease Time, minutes Lipase lipase Lipaselipase B. megaterium B subtz'lis N or =No readings taken.

Since the total time required for the germination phase of B. subtilisis 180 minutes (determined in the same manner as in Example I) and B.megaterium is 100 minutes (Example HI) it can be seen from the aboveTable 5 that the spores acquire heat sensitivity at a faster rate thansome of the other characteristic changes of germination. Also, it can beseen that by the time germination is complete, a substantial majority ofthe spores will not continue to develop during the outgrowth phase. Thisis also true when lipase and alkaline protease is used to germinate thespores as the total time for B. megaterium is 68 minutes and that for B.subtilz's is minutes (both times determine in the same manner as Example1).

EXAMPLE VI obtained:

TABLE 6 B. subtilis B. meqaterimn sublethal sublethal No heat, heat, Noheat, heat, minutes minutes minutes minutes As can be seen from Table 6,pretreatment with sublethal heat decreases germination times.

EXAMPLE VII 'In order to test the effectiveness of enzymes of thepresent invention against natural spore contamination in canned food,the following procedure is employed. 25 two ounce cans of slicedmushrooms are treated by separately injecting 5 ml. of enzyme lipase toprovide about 20,000 units per can and 10 mg. of glucose per can. Cansare sealed with solder and incubated along with a control, Le, 25 canscontaining no enzymes, sealed at 37 C. for three hours on a rotaryshaker. Cans are then subjected to a temperature of C. for 10 minutes inthe autoclave to inactivate the enzyme. Cans were allowed to cool andheld at 55 C. for 30 days after which time they were evaluated. Cans arethen determined to see if they have buckled or if they are swollen butnot buckled. Gases produced in those cans which is the cause forswelling and even bursting of cans. When these cans are opened, a verysour odor is noted and the pH has usually decreased from about 6.2 toabout 5.1. A direct microscopic observation of the liquor from theswollen cans reveals the presence of many long thin rods. Theseobservations are characteristics of thermophilic anaerobic spoilagefrequently encountered in canned mushrooms. At the end of the storageperiod, control and enzyme treated cans are opened. The enzymes had noobservable effect on the mushrooms and the overall quality of the enzymetreated mushrooms appeared to be as high as controlled mush rooms.

The same procedure is used with the exception that 25 cans of mushroomsare injected with a combination of lipase and a supplementary enzyme andalkaline protease. The combination is injected so that there are presentabout 20,000 units of lipase and about 70,000 units of activity ofalkaline protease per can.

EXAMPLE VIII The procedure of Example VI is followed with the exceptionthat a combination of lipase and a physiological germinant L-alanine isinjected into the mushroom cans. The combination is injected so that20,000 units of lipase are present and about 1 ml. of 0.1 M L-alanineare present per can of mushrooms.

EXAMPLE IX The procedure of Example VI is repeated except there issubstituted for the lipase a combination of lipase a supplementaryenzyme and a physiological germinant L-alanine. The combination isinjected into each can so that there are present 20,000 units ofactivity of lipase and 13 70,000 units of activity of alkaline proteaseand 1 ml. of 0.1 M the physiological germinant L-alanine.

It is to be understood that the following claims constitute part of thedescription of the present invention and consequently are to beconsidered as such.

What is claimed is:

1. A method for promoting the germination of spores of bacteria selectedfrom the group consisting of Bacillus, genera and Clostridium genera,which comprises contacting said spores with a lipase enzyme, said lipasebeing present to provide about units of activity per about 10 to about10 spores.

2. A method according to claim 1 wherein said spores to be germinatedare selected from the group consisting of Bacillus sublilis spores,Bacillus megaterium spores and Bacillus stearothermophilus spores.

3. A method according to claim 1 wherein said spores are germinated at atemperature of about 25 C. to about 50 C., and said lipase enzyme ispresent in an amount to provide about 800 units of activity to about30,000 units of activity per about 10 to about 10 spores.

4. A method according to claim 3 wherein said spores are in an aqueousmedium.

5. A method according to claim 1 wherein said spores before germinationare heated at a temperature of from about 40 C. to about 125 C. for aperiod of about 30 seconds to about 60 minutes.

6. A method according to claim 3 wherein said spores before germinationare heated at a temperature of about 40 C. to about 125 C. for a periodof about 30 seconds to about 60 minutes.

7. A method for promoting the germination of spores of organism selectedfrom the group consisting of Bacillus genera and Clostridium generawhich comprises contacting said spores with a lipase enzyme and with asupplemental enzyme selected from the group consisting of proteases,amylases, and phosphatates, said lipase being present to provide about10 units of activity per about 10 to about 10 spores.

8. A method according to claim 7 wherein said spores to be germinatedare selected from the group consisting of Bacillus megaterium, Bacillussubtilis and Bacillus stearothermophilus.

9. A method according to claim 7 wherein said spores are contacted withsaid lipase enzyme and said supplemental enzyme at a temperature ofabout 35 C. to about 45 C. wherein said lipase is present in an amountto provide about 800 units of activity to about 30,000 units of activityper 10 spores to about 10 spores and wherein said supplemental enzyme isan alkaline protease in an 1 4 amount to provide about 10,000 units ofactivity to about 500,000 units of activity, per 10 to about 10 spores.

10. A method according to claim 9 wherein said spores to be germinatedare contacted first with said lipase for at least 10 minutes, whencontacted with said alkaline protease.

11. A method according to claim 7 wherein said spores before germinationare heated at a temperature of from about 40 C. to about 125 C. for aperiod of about 30 seconds to about minutes.

12. A method for promoting the germination of spores of bacteriaselected from the group consisting of Bacillus genera and Clostridiumgenera which comprises contact ing said spores with a lipase enzyme anda physiological germinant, said lipase being present to provide 10 unitsof activity per about 10 to about 10 spores.

13. A method according to claim 12 wherein additionally said spores arecontacted with a supplemental enzyme selected from the group consistingof protease, phosphatases and amylases.

14. A method according to claim 13 wherein said spores beforegermination are heated at a temperature of about 40 C. to about C. for aperiod of about 30 seconds to about 60 minutes.

15. A method according to claim 12 wherein said spores to be germinatedare selected from the group consisting of Bacillus subtilis, Bacillusmegaterium and Bacillus steamthermophilus.

16. A method according to claim 15 wherein said spores are germinated ata temperature of about 25 C. to about 50 C., said lipase enzyme ispresent in an amount to provide about 800 units of activity to about30,000 units of activity per 10 spores to about 10 spores and saidphysiological germinant is selected from the group consisting ofL-alanine, inosine, glucose and mixtures thereof.

17. A method according to claim 16 wherein said spores are additionallycontacted with an alkaline protease supplemental enzyme, said enzymebeing present in an amount to provide about 10,000 units of activity toabout 500,000 units of activity per about 10 spores to about 10 spores.

References Cited UNITED STATES PATENTS 3,276,840 10/1966 Sierra -963,328,178 6/1967 Alderton 99-215 ALVIN E. TANENHOLTZ, Primary ExaminerUS. Cl. X.R.

